Around ten million people are infected with HTLV-1 worldwide, and 1–4% develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), characterized by an important degeneration of the spinal cord, which can lead to death. Distinct HLA alleles have been associated with either HAM/TSP susceptibility or protection. However, these HLA alleles set may change according to the population studied. Brazil is the second country in the number of HTLV-1-infected people and there are few reports addressing the HLA influence on HTLV-1 infection as well as on disease outcome. The objective of this study was to evaluate the influence of HLA alleles as a risk factor for HAM/TSP and the proviral load (PVL) levels, clinical progression, and death outcomes in an admixed Brazilian population. The HLA-A, -B, -C, and -DRB1 were genotyped in 375 unrelated HTLV-1-infected individuals divided into asymptomatic carriers (AC) (n = 165) and HAM/TSP (n = 210) in a longitudinal cohort from 8 to 22 years of follow-up. Because locus B deviated from Hardy–Weinberg Equilibrium for the study groups, the results represented for HLA-B alleles were inconclusive. The alleles HLA-A*68 and -C*07 were related to HAM/TSP risk in multivariate analysis. The alleles HLA-A*33, and -A*36 were associated with protection against disease progression in HAM/TSP patients, while -C*12, -C*14, and -DRB1*08 were associated with increased risk of death. In the AC group, the presence of, -C*06 and -DRB1*15 alleles influenced an increased PVL, in an adjusted linear regression model, while -A*30, -A*34, -C*06, -C*17 and -DRB1*09 alleles were associated with increased PVL in HAM/TSP group compared to HAM/TSP individuals not carrying these alleles. All these alleles were also related to increased PVL associated with clinical progression outcome. Increased PVL associated with the death outcome was linked to the presence of HLA-A*30. PVL has been associated with HLA, and several alleles were related in AC and HAM/TSP patients with or without interacting with clinical progression outcomes. Understanding the prognostic value of HLA in HAM/TSP pathogenesis can provide important biomarkers tools to improve clinical management and contribute to the discovery of new therapeutic interventions. Approximately 5 to 10 million people are infected with HTLV-1 worldwide4. Japan and Brazil are countries with the highest estimated total numbers of HTLV-1-infected people5. The HTLV-1 prevalence in Brazil ranges from 146.3 to 390.2/100,000 inhabitants4. Although most infected individuals remain asymptomatic, around 0.25–3.8% develop HAM/TSP over time6. This neurological disease is characterized as a slowly progressing spinal cord disease. The leg muscles gradually become weak. The limbs are stiff, the movements are slow, and walking becomes more difficult. Muscle spasms in the legs are common. Other HTLV-1-associated diseases, such as neurogenic bladder7 and ocular manifestations8, can also be present among HTLV-1-positive individuals. These clinical outcomes are associated with increased HTLV-1 proviral loads (PVL)9; however, it is still unclear what determines the high PVL in these patients. The feature that only a small percentage of HTLV-1 carriers will develop some symptoms has not yet been fully elucidated.The human T cell lymphotropic virus (HTLV-1) is a retrovirus associated with two primary diseases: adult T cell leukemia/lymphoma (ATLL)9.The neurological injury in HAM/TSP may be a consequence of an inflammatory reaction triggered by the recognition of infected cells by cytotoxic T lymphocytes, followed by the release of cytokines and damage to the central nervous system. Factors related to the HTLV-1/host interaction may be involved in the risk of developing HAM/TSPIn the search for a biomarker that can identify who is more prone to develop the disease, depending on the population's geographic distribution, some studies have shown that HLA alleles can influence the course of the infection.14. DRB1*01, in the absence of A*02 and C*08, was related to a higher risk of developing HAM/TSP in the Iranian population16. In addition, B*07 and DRB1*01 were related to an increased risk of developing HAM/TSP in Spain, where the majority of the individuals studied were from Latin America17. In South America, the allele B*35 was associated with a higher risk of developing HAM/TSP in Argentina18. On the other hand, the HLA-A*02, -C*08, -B*40, -DRB1*15, and -DQB1*06 were related to protection in Japan13. However, in Iran, Caribbean, Peruvian, and Spain populations, the same HLA alleles were unrelated to HAM/TSP susceptibility or protection22. Only two studies were performed in Brazil, involving a very small sample size of patients with HAM/TSP (n = 9) or selecting alleles previously associated with protection and disease risk in other geographic populations24. Unfortunately, these studies did not assess the complete HLA alleles or compare the clinical outcome. In addition, the Brazilian population is highly admixed and a probable association of ethnicity should be considered. Our objective was to describe the genetic polymorphism in the HLA-A, -B, -C, and -DRB1 loci in a subset of the Brazilian population infected with HTLV-1 in a longitudinal follow-up cohort at a Reference Center for Infectious Diseases in Rio de Janeiro, Brazil.The HLA alleles A*31, B*07, B*54, C*07, DRB1*01, and DQB1*05 were described to be related to a higher risk of developing HAM/TSP in Japanp = 0.14). The median clinical follow-up time was 13 , and 17 (14.29–16.57) years for HAM/TSP and AC, respectively. The distribution of gender and ethnicity ) was similar in both groups. Also, the HAM/TSP group presented a higher frequency of other clinical manifestations such as the neurogenic bladder (79.5%) and ophthalmic alterations (10.5% of the patients) compared to the AC group, p < 0.01. PVL was higher in the HAM/TSP group compared to the AC group (p < 0.01).Table p < 0.01). The clinical follow-up in the AC progressors  subgroup ranged from three to 26 years .During the clinical follow-up, 24 (11.4%) patients died from secondary causes related to HTLV-1 infection, such as recurrent urinary infections, bedsores, and sepsis. The most common causes of death among HAM/TSP in our cohort were urinary sepsis and deep venous thrombosis associated with pulmonary thromboembolism. All of these individuals were HAM/TSP patients. The clinical progression was investigated during the follow-up, and 148 (70.5%) HAM/TSP and only seven (4.2%) AC patients, showed a deteriorating outcome (p < 0.0001). The difference in clinical evolution between those individuals with HAM/TSP with unilateral support and bilateral support was significant (p = 0.0462); however, there was no difference in clinical development between individuals who needed unilateral support and those with an independent gait.Figure 25, updated to January 2023, except for the HLA-A*68 allele. From 5,585,608 HLA-typed Brazilians HLA-A*68 represented 6.20% and in the Rio de Janeiro State 6.41% among 254,519 donors, in contrast with 10.37% observed in our study population. Supplemental Table The frequencies of the HLA -A, -B, -C, and -DRB1 alleles in the study population were compatible with those found at the Brazilian Bone Marrow Volunteer Donor Registry (REDOME)p = 0.01, and OR 1.61 , p = 0.03, respectively), and were associated to HAM/TSP risk. As HLA-C*07 in the absence of -A*02 has been previously described as a risk factor for HAM/TSP24, we tested its association in the absence of the -A*02 allele. The contingency table analysis showed that the presence or absence of the -A*02 did not influence the -C*07 allele distribution in both groups.The B locus deviated from the Hardy–Weinberg Equilibrium (HWE) for both HAM/TSP and AC groups , -C*14 , p = 0.01) and -DRB1*08 , p = 0.01) were associated with death in HAM/TSP patients in the adjusted model , while 148 patients in HAM/TSP group showed clinical progression. Therefore, we only analyzed the association between HLA and disease progression in the HAM/TSP group Table . The all26 but not in other populations. We analyzed whether HLA alleles in our population alter the PVL. We performed an independent case and control analysis because PVL was higher in the HAM/TSP group compared to the AC group (Table p = 0.008) and -DRB1*15 , p = 0.012) had an increased PVL compared to those who do not carry those alleles , -A*34 , p = 0.005), -C*06 , p = 0.012), -C*17 , p = 0.03) and -DRB1*09 , p = 0.003) alleles had an increased PVL compared to those who do not carry those alleles , 27. This is a limitation of our case–control study. Some studies have associated HLA genetic polymorphism with protection or susceptibility to HAM/TSP development. This study evaluated the influence of HLA class I and II gene polymorphism in HTLV-1 infected individuals in a longitudinal follow-up and its association with PVL levels, clinical progression, and death outcomes. AC and HAM/TSP patients were accompanied for two to a maximum of 27 years, and clinical progression was related to HAM/TSP patients. The lifetime risk of HAM/TSP is different among ethnic groups26. Some studies report that it can take up to 21 years from the onset of infection to using a wheelchair in HAM/TSP28. Our study could not specify the median time patients became wheelchair users because the walking condition was only assessed in the first and the last clinical consultation. However, HAM/TSP patients that presented a clinical progression were in the cohort for at least eight years. Regarding mortality in HAM/TSP patients, it was pointed out that it would be a consequence of complications of the disease itself and co-infections with HCV/HIV29. Although there are some case reports of patients with HAM/TSP developing ATL30, no large-scale prospective cohort studies have been conducted on patients with HAM/TSP, nor has the risk of ATL transformation been evaluated in these patients. None of the patients included in the present study died of ATL.Brazil is a country with continental proportions and endemic to HTLV-1. Its population is composed of miscegenation among individuals of Indigenous, African, Asia, and European origin. Unfortunately, the Brazilian population is underrepresented in public genomic reference databases. Previous data comparing whole exome sequencing and SNP array from the Brazilian population and the 1000 Genomes datasets showed that there are significant differences in allele frequencies for both common and rare variants and this result indicates that none of the admixed American populations present in the 1000 Genomes dataset can be used as a surrogate for studies of the Brazilian population31. However, recently a review article discussed the role of HLA-B in 1322 Brazilians compared to different admixed populations worldwide32. These authors found that HLA-B gene diversity was higher in admixed and European populations while was lower in African populations. Studying gene diversity in admixed Brazilian population may increase the dataset in others genes banks around the world. As an example, 23 new alleles of the HLA-B gene were exclusively identified in the Brazilian population32. The high variability in the HLA-B gene must be studied in light of the role of their alleles in infectious diseases, so common in developing countries. Further studies should address the association of the HLA-B locus with HAM/TSP risk in a large admixed population to increase the likelihood of identifying the disease relationship.Unfortunately, both groups from our HTLV-1 study population were in HW disequilibrium for locus HLA-B, in opposition to the studied 2,624 renal transplant candidates from São Paulo State, Brazil34. The close relationship between HTLV-1 infection and uveitis has been identified concerning the ophthalmologic alterations, indicating that HTLV-1 is also highly associated with intraocular inflammatory disorder36. Some HLA alleles have been related to uveitis. The HLA-A*29 is strongly associated with Birdshot uveitis in individuals of Western-European ancestry, while HLA-B*27 is associated with uveitis, particularly in ankylosing spondylitis38. In our cohort, ophthalmic manifestations were more frequent in the HAM/TSP group, but it was not possible to correlate it with any HLA due to the small number of affected patients.In our cohort, PVL and neurogenic bladder were significantly higher in HAM/TSP than in the AC group, reinforcing what has already been published24. Borducchi et al.23 analyzed a small number of HTLV-1-infected patients (n = 71), including AC, ATLL, and HAM/TSP. No statistical difference was determined among these groups because of the small sample size. This study showed a tendency to a lower HLA-A2 frequency in HAM/TSP white patients and an association of HLA-DR11 with HAM/TSP only in the Mestizo patients, in disagreement with our study even after adjustment by ethnicity. Unfortunately, that report did not mention the number of HAM/TSP patients and did not compare their results using multivariable analysis. In another study24, only alleles previously associated with protection and disease risk were tested in a small cohort of 84 AC and 9 HAM/TSP patients. They identified that the alleles HLA-C*07 in the absence of -A*02 was associated with an increased risk of disease, and alleles C*08 and B*07 had no association with the development of HAM/TSP. In our study population, the presence or absence of A*02 did not influence the C*07 allele, and C*08 also showed no association with the disease.Only two studies with small sample sizes were carried out in Brazil to investigate the HLA association with HTLV-1 infection12, neither A*02 nor the HLA-C*08 alleles were associated with the protection for HAM/TSP development.In our study, the most frequent allele observed at locus A was A*02  and native Amerindians (7.32%) compared to white (5.66%) ones), the difference observed in patients with HTLV-1 was much more expressive (10.37%), and even greater in HAM/TSP patients (13.29%), suggesting a possible increased risk of this allele for HTLV-1 infection and disease progression in the Brazilian population. Indeed, in an HIV-positive African population, the allele -A*68 was associated with the rapid transmission of HIV in serum discordant heterosexual partners, with 21.3% of frequency39 and was associated with a protective role to SARS-COV-2 severe infection and death in a Mexican population, where this allele has a high frequency 40. HLA-A*68 is more frequently identified in South American indigenous populations, such as Argentina, Paraguay, and Chile43. Besides REDOME data there is a lack of reports describing the frequency of the HLA-A*68 allele in Brazil or in infectious diseases. More studies addressing the role of HLA-A*68 in the Brazilian population in the context of HTLV-1 infection might confirm his association with the augmented risk for infection and disease progression as observed in our study. The -C*07 allele was cited in the Japanese population as a risk factor for HAM/TSP13, as well as in our study. Interestedly, in an Argentine population, -C*07 was associated with an increased risk of ATLL but not with HAM/TSP18. This allele was underrepresented in AIDS Brazilian patients conferring protection against cytomegalovirus retinitis44.Our results showed that the alleles -A*68 and -C*07, had a higher frequency among HAM/TSP patients than in the AC individuals, leading to an increased risk of HAM/TSP. To our knowledge, no previous studies indicated the association of -A*68 with HAM/TSP. However, we should take into account that the -A*68 allele had a higher frequency in our study cohort when compared to the frequency of this allele in the REDOMEThis is the first study that analyzed whether HLA alleles could influence clinical progression in a longitudinal cohort. Patients were clinically followed for 20 years, and their walking clinical progression condition was determined. In our cohort -A*33 and -A*36 were associated with clinical progression and to our knowledge these alleles were not described in other populations. The role of these alleles should be clarified in other populations concerning their physiopathological properties.19. We identified new alleles that were associated with increased PVL in the AC group, carriers of the alleles -C*06, and -DRB1*15, and in the HAM/TSP group, carriers of the alleles -A*30, -A*34, -C*06, -C*17 and -DRB1*09. Although the presence of these alleles influenced an increased PVL, among these alleles, none of them was previously reported. Only B*40 was cited in a Japanese population study13, where its distribution was associated with a protective effect. In our cohort HLA-B alleles were in disequilibrium of HW in both cases and control groups.There is a clear association between HLA alleles and PVL levels13 and is cited with a tendency to a higher frequency in HAM/TSP patients than in HCs in a French Afro-Caribbean population22, but without association with PVL. It is the first time these associations are described in the Brazilian population.Further, we analyzed if the presence of the HLA alleles were responsible for the increase in PVL regarding the clinical progression outcome. In the AC group, carriers of the alleles: -C*06, and -DRB1*15 had an increased PVL. In the HAM/TSP group, the alleles -A*30, -A*34, -C*06, -C*17, and -DRB1*09 also influenced PVL and risk for clinical progression. Intriguingly, the alleles that influence PVL and clinical progression in the AC group are not the same as in the HAM/TSP group. Among these alleles, the only correlation with other studies we found in the -DRB1*15 allele, which in the Japanese study had a protective effectMoreover, we can highlight that the presence of the HLA-C*06 allele is associated with PVL, clinical progression, and death outcomes for the first time in our population. Studies carried out in ethnically different populations have not demonstrated equanimity in their results regarding risk or protection for the development of HAM/TSP. The host's genetic background must have an important role in this issue.45 and two previous reports from Brazilian cities  showed no association with sequenced viral genotype and clinical outcomes. In Brazil, Genotype a (Cosmopolitan) and subgroup A  is the most frequently (> 95%) identified HTLV-1 genotype48. None of our enrolled patients was Japanese or their descendant, in which Japanese subgroup B could be identified.HTLV-1 has remarkable genetic stability50. Furthermore, Brazil is the large population in Latin America and the fifth-largest in the world, and the Brazilian population is underrepresented in public genomic reference databases27. In these databases, 80% of all genetic data is related to the European ancestry population which accounts for only 16% of the world´s population. This gap prevents the discovery of new rare allele variants. Few studies reported the ancestry background of the general Brazilian population51 and studies associating ancestry analysis and HLA frequencies could better relate the role of HLA polymorphisms in the HTLV-1 infection and its medical importance. Furthermore, the study was conducted with clinical data collected retrospectively in a convenience sample. It was impossible to match patients with similar and long clinical follow-up times and with similar disease stages. Another limitation of this study was the reduction of the HLA typing to the level of allelic group. Although we have used in part of the typing a medium-level resolution that could result in better discrimination of the HLA alleles of risk and susceptibility, we were also limited by the HLA frequencies that tend to be small and implying having a larger cohort to have statistical differences. Nonetheless, we are not aware that allelic level should be the final goal for HLA studies, as in many diseases that initially were associated with an antigen  that with further studies have pointed out that only some alleles of the group were in association with the disease, as for spondyloarthropathies with HLA-B*2752 or rheumatic arthritis HLA-DRB*0453. Our results show an association between HLA frequencies and HAM/TSP risk, PVL, disease progression, and death. Because of the low number of participants presenting some HLA alleles, the results observed in this work have to be confirmed in other populations. Nevertheless, in light of our acknowledgment, this is the study involving the highest number of participants associating HLA alleles and HTLV-1 infection in the Brazilian population. Future studies, designed with an even greater number of patients, with a long and similar clinical follow-up period, and which include patients in the early stages of the disease, may increase the strength of comparisons between some HLAs regarding their role in HTLV-1 infection in the Brazilian population, reinforcing our findings.A major limitation of our study is related to the Brazilian genetic background. Brazilian population, as well as all Latin American countries, was composed through centuries of European colonization and the African slave trade. The continuous admixture events among Europeans, Native Americans, and West-Central and Southeast Africans occurred differently in each region of the country, producing a stratified population, even in the current days. Despite our study population showed similar HLA frequencies compared to REDOME , it may not reflect the entire genetic diversity of the Brazilian population. Even though, the Brazilian population has a large proportion of European white populationThe HLA system is vital in response to infections. In the case of HTLV-1 infection, where a minority of individuals carrying the virus will develop the neurological disease, the genetic determinants could be seen as an interplay between different immunological events. However, more studies should be conducted to monitor the HLA profile in AC patients at risk of developing the disease in longitudinal follow-up.In this study, we investigated the influence of HLA polymorphisms on the susceptibility for the development of HAM/TSP and clinical progression in a longitudinal cohort of an admixed Brazilian population. We found that HLA-A*68 and -C*07 carriers presented an augmented risk for HAM/TSP development in HTLV-1-infected individuals. In addition, -C*12, -C*14, and -DRB1*08 were associated with an augmented risk of death in HAM/TSP individuals. In contrast, the alleles HLA-A*33, and -A*36 were related to protection against disease progression in these individuals. We also showed that asymptomatic -C*06 and -DRB1*15 carriers have increased proviral load, while -A*30, -A*34, -C*06, -C*17, and -DRB1*09 alleles were associated with this characteristic on HAM/TSP patients. We also demonstrated that patients with high PVL and carrying these alleles presented disease clinical progression, while the presence of HLA-A*30 in these patients was associated with death. This is the first study to evaluate the influence of HLA alleles on the risk for HAM/TSP development as well as in clinical progression and death, in an admixed Brazilian population. Brazil has one of the highest rates of HTLV-1 infection, and studies addressing genetic factors associated with HAM/TSP risk could prevent disease worsening through earlier medical care.The Laboratory for Clinical Research in Neuroinfections at the Evandro Chagas National Institute of Infectious Diseases (Lapclin-Neuro), FIOCRUZ (INI/Fiocruz), Rio de Janeiro, RJ, Brazil, is a reference center for diagnostic and clinical follow-up of asymptomatic carriers and patients with neurological manifestations of HTLV-1 infection. Since 1999 more than 1000 AC and neurological patients, the majority of them with HAM/TSP, have been investigated and followed up in a longitudinal cohort by a team of neurologists. According to their clinical visit, HTLV-1 positive and genetically unrelated patients were randomly and consecutively included from the longitudinal study cohort. The Research Ethics Committee of the INI/FIOCRUZ (CAAE-0012.0.009.000-08) approved this study, and all subjects provided written informed consent. Volunteers were consecutively followed from 1999 to 2020 in a longitudinal cohort study from eight to 22 years. A sex, age, and ethnicity-matched unrelated case–control study was then designed to identify HLA's influence on the disease disability status, PVL, disease progression, and death outcomes. All clinical data were collected retrospectively, using a convenience sample provided by the cohort follow-up. A comprehensive medical history relating to neurological illnesses was taken at the time of inclusion in the cohort and annually for the AC patients and biannually for the HAM/TSP patients.54. Patients’ inclusion criteria were defined by positive serology for HTLV-1 in the blood (for AC) and blood and cerebrospinal fluid (CSF) for neurological patients . The screening for HTLV-1/2 infection was performed throughout the serological test using a Recombinant ELISA HTLV1/2 kit from Wiener , and confirmation of HTLV-1 infection through western blot analysis . Patients with HTLV-2 infection were not enrolled in the study. Individuals were divided into two groups: individuals with HAM/TSP, and other isolated neurological findings such as isolated neurogenic bladder, and HTLV-1 asymptomatic carriers (AC). The HAM/TSP participants were classified according to their disability status using the IPEC Clinical Disability Scale (IPEC-CDS) as described elsewhere55. The IPEC disability scale is a numerical scale ranging from 0 (no disability) to 29 . This scale is a composite of motor, pain, sensory, and sphincter subscores55.The clinical assessment and classification of the neurological cases followed the World Health Organization guidelines and diagnostic criteria for HAM/TSPClinical progression was determined by comparing patients' gait performance according to the IPEC-CDS motor subscore at the first clinical visit, when patients were included in the cohort, with the same score obtained at the last clinical visit. Any worsening in the IPEC-CDS motor score was considered disease progression, regardless of the initial score assigned to patients at the time of study enrollment. We also did not consider the degree of progression, meaning that patients who progressed to two or more levels on the motor scale were not separated from those who progressed to only one level.A survival curve was generated to analyze the longitudinal clinical progression in HAM/TSP patients, using each of their initial clinical classifications regarding the ability to walk . A change in the motor scale of worsening prognosis was considered a sign of disease progression.Peripheral blood (5 mL) was collected in EDTA tubes from all individuals, and DNA was extracted using the Puregene commercial kit , following the manufacturer’s instructions.First, the HTLV-1 cohort was HLA genotyped through an in-house PCR-SSP (polymerase chain reaction-sequence-specific primer) from 1999 to 2007 (n = 273), and throughout PCR-SSO (polymerase chain reaction with specific oligonucleotide probes) technique (n = 102).56. Primers were donated by the Department of Immunology at the St Mary’s School of Medicine, Imperial College, London, UK, and the PCR-SSO technique, using DNA LABType® trading system  was performed, according to manufacturer`s instructions, that provides SSO probes for the sequence-specific oligonucleotide linked to fluorescent microspheres for the identification of HLA alleles in genomic DNA samples.The HLA class I loci  genotyping was performed in an in-house PCR-SSP according to the methodology established by the Organ Transplant Laboratory from Oxford University4 cells was calculated by the following equation: [(copy number of tax gene)/ (copy number of β-globin gene/2)] × 10,00033. The mean of proviral load measures during the follow-up was used to determine the proviral load of each patient.The HTLV-1 PVL DNA was measured by real-time PCR assay  using the TaqMan system (Applied Biosystems) to amplify a 159 bp fragment of tax gene with SK43 and SK44 primers. A standard curve was generated using the β-globin gene as a reference. DNA from the TARL-2 cell line, which contains a single copy of the provirus HTLV-1, was used to establish the standard curve for tax gene quantification. The proviral load (PVL) in 10p-value < 0.05 was considered significant. All analysis was performed using R software version 3.6.3.A descriptive analysis of clinical and demographic characteristics was performed. Outcomes  and alleles  were associated using absolute and relative frequencies or mean and standard deviation. Hardy–Weinberg equilibrium was calculated by Exact test using a Markov chain to define the equilibrium in the allelic distribution in HLA-A, -B, -C, and -DR locus between HAM/TSP and AC patients. Prism 7 was used to determine a survival curve to analyze the walking ability's clinical progression. The Mann–Whitney U test evaluated quantitative variables, whereas the univariate Odds Ratio (OR) was used for categorical variables. We used a multivariate generalized linear Binomial model to estimate OR and Ci = Is. Linear models were tested to estimate the average difference in PVL among alleles and outcomes. A All research was performed following relevant guidelines/regulations described by the policies of the Nature Portfolio journals, and the Declaration of Helsinki. This study was approved by the Research Ethics Committee of the INI/FIOCRUZ (CAAE-0012.0.009.000-08) and all participants provided written informed consent.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


This study aimed at examining an eight-week moderate-intensity comprehensive exercise training program on the parameters of sarcopenia in elderly females.A total of 49 community-dwelling elderly females with sarcopenia (65.5 ± 2.5) were assigned randomly to an experiment group  and a control group . In the EG, an eight-week comprehensive exercise training program was implemented, in 1 h, 3 times per week, a total of 24 sessions. The CG only received health public education per two weeks, a total of 4 times. Subsequently, the differences between the two groups were tested through two-way repeated ANOVA.2, respectively. Group-by-time interactions were significantly different on the ASM  and SMI . Muscle strength was improved 0.8 kg in the EG. Significant group-by-time interaction differences were reported in the handgrip strength  after the eight-week intervention. Compared with the baseline, gait speed was improved a 0.05 m/s and 5-time chair stand was decreased a 0.27 s in the EG. Group-by-time interactions were significantly different in 5-time chair stand .ASM, SMM, and SMI in the EG were significantly improved by 0.26 kg, 0.18 kg, and 0.10 kg/mThe moderate-intensity comprehensive exercise was confirmed as a safe and convenient exercise program. Although a load of training intensity is not sufficient to improve the gait speed, this exercise protocol is promising in delaying overall results in community-dwelling sarcopenia elderly females and contributes to the improvement of muscle mass, handgrip strength, and 5TCS. This agAs a progressive and skeletal muscle disease, sarcopenia is associated with an increase in negative consequences such as falls, disability, and lower quality of life in the elderly. The importance of preventing and managing sarcopenia is self-evident. Until now, there are no approved drugs for the treatment of sarcopenia, nutritional supplements, and exercise have received attention as a possible non-drug treatment for the prevention or treatment of sarcopenia. Protein intake is an important stimulant of muscle synthesis. However, studies showed that 15–38% of older men and 27–41% of older women consume less than the recommended daily allowance of protein . These pIt is urgent and necessary to pay attention to the improvement of physical performance in the elderly and delay the progression to severe sarcopenia. There are inconsistencies in the effectiveness of exercise interventions in improving physical performance in the elderly with sarcopenia. Kim  and Seo 22.1A prospective, dual-arm parallel design, community-based study was conducted. This study gained approval from the Institutional Review Board of the University of Ulsan (1040968-A-2022-005) and Henan University of Science and Technology (2022–0180).The participants of this study were assigned to an experimental group (EG) receiving an eight-week moderate-intensity comprehensive exercise and a control group (CG), which was highlighted not to receive any exercise or nutrition program but only health education for 4 times. In accordance with the ethical standards of the Declaration of Helsinki, all participants were presented the detailed contents of the exercise intervention and then signed informed consent.2.2The subjects were recruited from the District of Jian Xi, Luoyang by posters, flyers, and WeChat. In accordance with the AWGS 2019 consensus, sarcopenia-associated parameters of 96 participants were examined to illustrate whether they conformed to the sarcopenia diagnosis criteria. The additional inclusion criteria comprised (a) participants aged ≥60 years, (b) independence ability , (c) no experience in a supervised exercise for three months prior to research. Any of the settings were excluded, comprising (a) diabetes mellitus, (b) cardiovascular diseases, uncontrolled hypertension (>140/90 mmHg), (c) gallstones, (d) kidney stone, (e) infectious diseases, (f) upper/lower extremity fracture, (g) knee or hip prosthesis, (h) pacemaker, (i) musculoskeletal disorders, (j) psychiatric disorders, (k) epilepsy, (l) any medical treatment that affects sarcopenia outcomes, (m) cognitive impairment ; (o) physical dysfunctions.2.3We performed an a prior power analysis using G*Power 3.1  for the 2.4The participants were asked to offer written informed consent after understanding the objective, rights, and potential risks. One trained researcher (CYZ) estimated the participants’ baseline characteristics. Then, an independent researcher (SSH) used a computer-generated list of random numbers  to rando2.5This comprehensive program takes on critical significance in muscle function, and physical performance . The respective group was administrated and guided by trained exercise specialists to ensure adherence to this protocol. The respective exercise session comprised nearly 60 min, three times per week for eight weeks, mainly including a 5-min warming-up, followed by a training exercise part of 45–50 min (combined simplified 24-form Tai Chi and progressive resistance training) in a broad and well-cool indoor activity room with a group-based setting, and then a 5-min cool-down part. The exercise program was conducted at a moderate intensity roughly 12–14 on the Borg rate of perceived exertion scale (Borg RPE). The experiment group was part into four subgroups to guarantee participants the appropriate and convenient instruction. All the participants practiced within their allocated subgroup.2.5.1Tai Chi might be benefit for improving balance, reducing falls and injury-related falls in older people , and imp2.5.2The progressive resistance exercise program was performed using elastic bands. Different colors of the above-mentioned elastic bands represented different resistance levels, with nearly a 20% resistance intensity difference between the respective color . To be specific, two days before the exercise intervention and one day before the fifth week the tests were performed to predict the respective participant's one-repetition maximum (1RM) in first-fourth and fifth-eighth weeks through a submaximal 60–80% RM test. In the first-fourth weeks, the exercise protocol was the same, the intensity varied every two weeks, as well as in the fifth-eighth weeks. However, some difference was identified between the first-fourth and fifth-eighth weeks, in which the training content and the repetition/set were modified. Furthermore, since the participants were the elderly, nearly a 45-s to 90-s rest was provided prior to the next part of the content.In terms of the actual sessions, participants were instructed and practiced four movements. The participants were guided to practice the respective movement at 40% of 1RM intensity using a yellow TheraBand initially first, and then the 1RM intensity was progressively increased to 60–80% with the adjustment to two-three sets of 8–12 repetitions. To facilitate the development of muscle function in the upper and lower body, participants were highlighted to do the respective concentric movement ‘as soon as possible’ and perform the eccentric movement in a relaxed and controlled status. Higher-intensity resistance was enhanced when the participants showed the capability of maintaining their RPE to a “somewhat hard” grade. However, if participants could not accept the next progressive intensity , the previous resistance intensity of the elastic band was maintained for an additional session until the participants could receive the next elastic band intensity color. The planning and implementation of the protocol were in accordance with the guidelines supported by the American College of Sports Medicine.2.5.3Participants in the CG will not be subjected to any recommendations about altering their daily diet or lifestyle during the eight weeks. They will receive calls or interviews once a week, and any health problem information was written down by a trained researcher. Meanwhile, health education was prescribed to them per two weeks. The topics of health education included primary healthcare skills, cognitive ability, etc., but were not shared with exercise or nutrition. CG subjects also received the same pre- and post-test as EG participants.2.62.6.12 in females.Height and weight were estimated by a height and weight machine  to the nearest 0.1 cm and 0.1 kg, respectively, without shoes. SMM and ASM were estimated according to BIA , and the2.6.2Handgrip strength is considered a feasible and convenient measure of measuring muscle strength for use in hospital services, clinical research, and primary health care settings . AWGS 202.6.3AWGS 2019 recommends that either one of the SPPB, gait speed  or 5-time chair stand test lower than the cut-off threshold could be defined as low physical performance , SMI . Significant interactions were examined with tests of simple effects. The simple effect test showed that the EG was significantly higher in sarcopenia index at W8 than the CG, with a 0.15 kg/m2 improvement (p < 0.01). In addition, the EG reported a significantly higher ASM and sarcopenia index at W8 than at W0. There was no significantly difference recorded in the CG at W8 than at W0.As shown in 3.22 = 0.13]. Significant interactions were examined with tests of simple effects. The simple effect test showed that the EG was significantly higher effect  at W8 compared to the CG. In addition, at W8, the EG reported a significantly 0.88 kg (p < 0.01) higher recording than at W0. On the other hand, the CG were no significantly difference than at W0, see The two-way interaction of groups and time were significant for handgrip strength . Significant interactions were examined with tests of simple effects. The simple effect test showed that the EG and the CG had a 0.42s (p < 0.01) difference between groups at W8. No statistical difference was identified in the gait speed and 5TCS at W8 in the CG, see At week 8, The EG showed a significant difference in gait speed, with a 0.05 m/s (p < 0.01) improvement, and 5TCS was reduced by 0.27 s (p < 0.01) to baseline. The two-way interaction of groups and time were significant for 5-time chair stand [F  = 6.35, η4This study confirmed the impact of an 8- week moderate-intensity comprehensive exercise protocol (combined Tai Chi and TheraBand progressive resistance training) on sarcopenia-associated parameters in community-dwelling elderly female sarcopenia. The primary finding is the eight-week moderate-intensity comprehensive exercise program improved 5TCS in elderly females with sarcopenia, but there is no significant difference recorded in the gait speed. The second finding is muscle mass, handgrip strength had a positive change after this eight-week comprehensive exercise intervention. Thus, the sarcopenia-associated outcomes (without gait speed) in elderly females with sarcopenia occurred beneficial change after eight weeks of the comprehensive exercise protocol.2). Generally speaking, skeletal muscle mass represents a crucial of strength, endurance, and physical performance. A previous 24-week combined resistance exercise and Yi Jin Jing had a positive effect on increasing skeletal muscle mass and delaying sarcopenia in the elderly [The present study presents evidence to support the benefits of exercise on muscle mass . Furthermore, more intensity subgroups should be separated to determine the optimal frequency/time. Lastly, mental health has taken rising significance in the sarcopenia elderly, and more studies should be conducted to analyze the effects of exercise on their satisfaction, self-efficacy, or quality of life.5The sarcopenia elderly should participate in an appropriate type of exercise program during non-pharmacological interventions to relieve or transform their sarcopenia status for better health and quality of life. The present moderate-intensity comprehensive exercise refers to a combination of aerobic, balance (simple 24-form Tai Chi) and resistance, and flexibility exercise (TheraBand exercise) in one exercise program strategy, i.e., a safe and convenient exercise program.Although a load of training intensity is not sufficient to improve the gait speed, this exercise protocol is promising in delaying overall results in community-dwelling sarcopenia elderly females and contributes to the improvement of muscle mass, handgrip strength, and 5TCS. Thus, community staff or sports instructors are recommended to receive the training for implementing this comprehensive exercise protocol program for the community-dwelling elderly with sarcopenia.Chen Bo-yuan: Conceived and designed the experiments; Performed the experiments; Wrote the paper.Shin So-hee: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Jie Chun-yang: Contributed reagents, materials, analysis tools or data; Wrote the paper.Chen Yuan-zhe, Chang Zhi-liang and Ding Hui: Performed the experiments; Analyzed and interpreted the data.Yang Hong: Performed the experiments.Data included in article/supp. Material/referenced in article.No additional information is available for this paper.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The Pulmonary Embolism Quality of Life (PEmb-QoL) questionnaire is the first disease-specific scale for assessing the quality of life in patients with a history of pulmonary embolism (PE).To assess the cross-cultural validity and reliability of the disease-specific PEmb-QoL questionnaire.The Persian version was prepared through the forward and backward translation of the English questionnaire. Six months after the diagnosis of acute PE, consecutive Persian-speaking patients were asked to complete the PEmb-QoL, the generic 36-item Short Form (SF-36) questionnaires and undertake a 6-minute walk test (6MWT). Acceptability was assessed via item missing rate, reproducibility by the test-retest method, and internal consistency reliability by Cronbach’s α and McDonald’s ω coefficients. Convergence validity was assessed using the Spearman rank correlation between scores of PEmb-QoL, SF-36, and 6MWT. The questionnaire structure was evaluated through exploratory factor analysis.Ninety-six patients with a confirmed diagnosis of PE completed the questionnaires. The Persian version of PEmb-QoL had good internal consistency , inter-item correlation (0.3–0.62), item-total correlation (0.38–0.71), reproducibility (test-retest ICC with 25 participants = 0.92–0.99), and good discriminant validity. Convergence validity was confirmed by the moderate-to-high correlations between PEmb-QoL and SF-36 scores, and a good correlation between the “limitation in daily activities” dimension of the PEmb-QoL questionnaire and 6MWT results. Exploratory factor analysis suggested a 3-component structure with functional , symptoms , and emotional  components.The Persian version of the PEmb-QoL questionnaire is valid and reliable for measuring the disease-specific quality of life in patients with PE.  •Pulmonary embolism (PE) has been shown to impact different aspects of the quality of life (QoL).•PE quality of life (PEmb-QoL) is a disease-specific survey for patients with PE.•Cross-cultural validity and reliability of PEmb-QoL were confirmed in a Persian-speaking cohort.•Minor adjustments in dimensions and their constituent items were required. AlthougPrevious statements highlight that the chronic complications actually experienced following acute PE go well beyond the implications of clinically measured outcomes such as RV dysfunction and pulmonary hypertension ,9,10. ThHealth-related quality of life is a multidimensional construct encompassing one’s self-perception of, at a minimum, physical, emotional, and social well-being . It is uPulmonary embolism quality of life (PEmb-QoL) questionnaire is the first and currently the only disease-specific PRO instrument for patients with a history of PE. The questionnaire was developed in Dutch, cross-culturally validated in several other languages, and is part of the recently developed core set of outcome measures for patients with PE . Our stu2Our study aimed to prepare the Persian version of PEmb-QoL based on the English version and then evaluate its psychometric properties  as to whether it is an appropriate instrument for measuring PE-related quality of life.2.1All Persian-speaking surviving patients with records in the Pulmonary Emboli Registry of Rajaie Cardiovascular Medical and Research Center (RHC-PE) between September 2015 and August 2018 were invited via phone calls to participate in the present prospective study. The RHC-PE registry is an all-comers cohort of patients with a confirmed diagnosis of PE . Briefly2.2Each patient was scheduled for a structural 6-month follow-up program composed of detailed transthoracic echocardiography and a 6-minute walk test (6MWT). During the program, a QOL assessment was performed using the disease-specific PEmb-QoL and the generic 36-item Short Form (SF-36) survey over an interview session assisted by an experienced nurse.2.32.3.1SF-36 is a widely used, generic, health-related QOL questionnaire validated in many disease cohorts and languages, including Persian . The que2.3.2Modeled based on the generic SF-36 and the disease-specific quality of life after acute DVT (VEINES-QOL/Sym) questionnaire, the PEmb-QoL was designed to assess disease-specific health-related QOL in patients with a history of acute PE . It cont2.4A cardiovascular disease specialist proficient in both English and Persian-translated the validated English questionnaire into Persian (forward translation). The translation was converted back into English (backward translation) by 2 different cardiovascular disease specialists proficient in both languages without reviewing the original English version. Afterward, 5 independent experts in pulmonary vascular diseases reviewed and compared the Persian and English versions to ensure clarity and to address any inconsistencies. As the final step, minor adjustments were made to the preliminary translation based on a pilot study in which 20 native Persian-speaking patients were interviewed regarding the clarity of the items after they had completed the first questionnaire draft.2.5Psychometric analysis was conducted according to the latest standard protocols .2.6Acceptability was assessed through the completeness of data (the missing item rate). Reliability was assessed in terms of consistency between the results of repeated measurements (reproducibility) and consistency between responses within the same measurement . Reproducibility was reported as an intraclass correlation via the test-retest method, where the respondents were invited back to our center 3 weeks after the initial QOL assessment to retake the PEmb-QoL questionnaire. The 3-week interval was selected to prevent recall and minimize the chance of clinical changes.Cronbach’s α is considered an adequate measure of internal consistency for individual dimensions . Still, The score distribution, along with the floor effect and the ceiling effect, was reported for the dimensions. The quality criteria for acceptability and reliability are summarized in 2.7The content validity of the PEmb-QoL has been previously investigated with respect to measurement aims, concepts, target population, and item selection ,39.In the present study, construct validity was investigated in terms of convergent and discriminant validity. A PRO measure is expected to have relatively high correlations with other theoretically similar PRO and non-PRO measures  while having relatively lower correlations with theoretically dissimilar measures  ,40. ThusWe further evaluated internal consistency, item selection, and questionnaire structure through exploratory factor analysis (EFA). EFA investigates the coherence of item responses to suggest an underlying structure for the questionnaire. We first used the Scree test to determine the appropriate number of latent factors  and then conducted EFA via a polychoric correlation matrix with the maximum likelihood estimation method. The polychoric correlation matrix was chosen over the conventionally used Pearson correlation, given that items with fewer than 5 to 7 response options  could violate the linearity assumption of the Pearson correlation . FinallyThe R programming statistical software version 4.1.3 was used with the tidyverse, Psych, GPArotation, ggcorrplot, and Ggally packages.33.1Of 170 patients recruited in the RHC-PE registry, 98 agreed to complete the PEmb-QoL questionnaire. Two participants were excluded as they had responded to fewer than 75% of the items. The responses elicited from the remaining 96 participants had a minimal item missing rate (0.38%), with 6 participants having a total of 14 unresponded items. The details of the patient flow and exclusion criteria are depicted in 3.2r = 0.43) and, expectedly, the strongest between limitations in the activities of daily living limitations and work-related problems (r = 0.75) as well as between the frequency of complaints and the intensity of complaints (r = 0.76) . Internal reliability was high (α ≥ 0.81) for almost all the dimensions except the intensity of complaints (α = 0.6). All dimensions had appropriate mean-corrected item-total correlations, ranging between 0.43 and 0.71 . Moreove = 0.76) .Table 2Ar = 0.34). This item was also the only item that, upon removal, would improve the internal consistency of its designated dimension (α = 0.83 to α = 0.85).P value = .02) and had more odds of being male . The test-retest analysis indicated moderate-to-high intraclass correlations for all dimensions, ranging from 0.70 for work-related problems to 0.97 for the intensity of complaints  returned to complete the questionnaire for the second time. Compared to the primary respondent group, participants of the retest were, on average younger  was excluded from the EFA as the majority of the participants (65%) had chosen the “I do not work” response. The EFA yielded 3 latent factors, cumulatively accounting for 60% of the total observed variances . Factor r = 0.47) to mental health (r = 0.58), which resulted in a roughly similar association with mental health (r = 0.64) and physical health summary scores (r = 0.63) of the SF-36. The median (IQR) distance walked during the 6MWT was 390 meters (320–450 m). Limited exercise capacity, defined as distances less than 350 meters, was observed in 39% of the participants. Expectedly, the 6MWT distance converged best with the scores from limitations in the activities of daily living (r = −0.61) and work-related problems (r = −0.35).The details of convergent and discriminant validity are presented in 4To our knowledge, The present study is the first, to translate and validate a PE-specific PRO instrument in a Middle Eastern country. Cross-cultural validation of the PEmb-QoL questionnaires in Persian-speaking populations would allow researchers to study the impact of PE and its treatments on the quality of life of a large population of PE survivors, given that over 120 million people worldwide speak the Persian language. Based on our results, almost all the dimensions proposed by the original validation study  yielded Overall, the PEmb-QoL total summary scale and emotional complaints conformed to almost all gold reliability standards, and their appropriate inter-item correlations and absence of ceiling or floor effects indicated their adequate discriminative power. Still, 5 out of the 6 dimensions had floor effects, with work-related problems also exhibiting a ceiling effect. Similar findings have been observed in all other validation studies of the PEmb-QoL questionnaire . These fAfter clarifying content validity in terms of measured concepts, Klok et al.  structurr = 0.38). The removal of item 1a is supported by the fact that the presented description of “pain behind or between the shoulder blades” is not only an uncommon, chronic complication of PE, but it could also overlap with musculoskeletal complaints [As suggested by the FDA guidelines , we compmplaints ,47. FourMazdak Tavoly et al.  was the P value = 0.03) and the fact that 12 patients died prior to study enrollment indicate that older and probably sicker patients may have been underrepresented in our study population. We believe this low turnout to be the result of our preferred mode of questionnaire administration; during their 6 months follow-up session, patients were asked to complete the questionnaires in the presence of an experienced nurse to minimize missing item responses, and accommodate low literacy, visually impaired, and elderly patients [Our study is subject to several limitations. First, of the 170 registered patients in the RHC-PE registry, only 98 (58%) participated in the PEmb-QoL evaluation . Of thespatients . An in-pIn summary, the Persian version of the PEmb-QoL questionnaire is a psychometrically valid and reliable PRO instrument to assess health-related QOL in patients with a history of acute PE. We extracted a 3-component structure comprising functional , symptom , and emotional  components. Further longitudinal studies are warranted to evaluate responsiveness and establish the minimum important difference.


Human T-lymphotropic virus (HTLV) infection has a worldwide distribution and currently, more than 2.5 million individuals have been infected in Brazil. The study aimed to investigate HTLV infection prevalence among blood donors in Mato Grosso do Sul, characterizing seroepidemiological profiles of HTLV-1/2 positive individuals and evaluating the blood bank's HTLV screening system. A cross-sectional survey was conducted among blood donors from Mato Grosso do Sul state (MS)—Central Brazil, between January to December 2021. The information was obtained from databases, samples from the collection of HEMOSUL, and active searching, with the completion of laboratory analyses. 35,278 blood donors were screened for anti-HTLV-1/2 by chemiluminescence immunoassay (CMIA). Among them, 78 were initially reactive for anti-HTLV-1/2 (2.21/1000). Out of 78, 67 returned to the blood center to collect a second sample for retesting with a second screening with CMIA. After confirmation, 8 samples were indeterminate, and 8 were confirmed as positive for HTLV antibodies. New tests were performed for the 8 positive samples, and 6 were confirmed as HTLV-1 infection , one as negative, and one as indeterminate. The present study describes the low prevalence of HTLV infection in blood donors from MS and contributes to the definition of the regional infection profile. The prevalence found in this study (0.017%–0.17/1000) shows to be a much lower value than the rates reported in other states in Brazil. We highlight the need for confirmatory testing for those seropositive donors in screening assays and the need for adequate counseling and patient management for those confirmed HTLV individuals. HTLV-1 is mostly associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy (HAM) but is also associated with other inflammatory diseases6. HTLV-1 has a worldwide distribution, with South Japan, sub-Saharan Africa, Caribbean Islands, Melanesia, and South America considered the most prevalent regions7.Human T-lymphotropic virus (HTLV) is a retrovirus with a tropism for T lymphocytes. HTLV-1 and HTLV-2 were identified in 1980 and 1982, respectively, besides HTLV-3 and HTLV-4 in 20058. From the data obtained, several seroprevalence studies were carried out. It is estimated that 2.5 million individuals are infected with HTLV-1 in Brazil, and a heterogeneous prevalence was observed according to the region and specific populations9. Previous studies conducted in the state of Mato Grosso do Sul  have reported rates varying from 0.1%—10.0% in specific groups, such as pregnant women10, indigenous population11, prisoners12, Japanese community14, men who have sex with men15 and remnant quilombos16.Considering that blood transfusion can be a route for HTLV transmission, screening of blood donors is important to prevent it. Thereat, screening for HTLV-1/2 infection in Brazilian blood banks has become mandatory since 1993This cross-sectional study aimed to describe the HTLV infection prevalence in blood donors in Mato Grosso do Sul state and the seroepidemiological profile of the positive HTLV-1/2 individuals, and also evaluate the screening system for HTLV of the blood bank.A total of 45,751 blood donations were screened between January 2021 to December 2021, corresponding to 35,278 blood donors. The general profile of blood donor candidates based on self-report before the donation was in majority men (55.48%), with ages ranging from 16 to 69 years (average 35 years). Most donors reported being widows (47.62%), 85.03% identify as Brazilian caucasian, and most donations were realized in the capital—Campo Grande (77.7%).Seventy-eight candidates were considered inapt due to being positively screened for anti-HTLV-1/2 , 67 returned to the blood center to collect a second sample for retesting with a second screening with CMIA. The spread of signal/cut-off ratio (S/CO) values of the second collection samples is presented in Fig. .21/1000,Of all 67 samples, 8 were indeterminate, and 8 were positive in confirmatory tests HTLV-1/2 INNO-LIA) realized in HEMOSUL  from HEMOSUL were retested by our group and were confirmed negative for HTLV infection. New tests were performed for the 8 positive samples using stored blood samples from HEMOSUL or through new blood collection of the 3 individuals who agreed to participate in the study. Of these 8 samples, 6 were confirmed as HTLV-1 , one as negative  (DMS-01: S/CO ratio 5.93), and one as indeterminate (S/CO ratio 50.56).10. The youngest child (DMS-02.01) was born in 2008 and breastfed for 2 years. Both DMS-02 and DMS-02.01 had blood collected by our research group. DMS-02 was confirmed for anti-HTLV-1, and DMS-02-01 had a negative result for anti-HTLV-1/2. The participant was referred to the medical service but did not attend and was going to a city change.One of the individuals who agreed to participate in the study (DMS-02) was a 46-year-old female and never donated blood before. She reported being married once when she had 2 children, and 2 cesarean surgeries were performed. Also, she reported never having a blood transfusion, having piercings or tattoos, having an accident with others’ blood, being arrested, or using drugs. She was unaware of having other IST. No information was available on the HTLV-1 status of her ex-husband. The first child was born in 2006. At that time, MS state had already been testing anti-HTLV on antenatal since 2002The other positive (DMS-04) was already a blood donor, a 40-year-old female, and married twice. No information was available on the HTLV-1 status of the first husband with whom she had her first child in 2002, and she reported breastfeeding for 9 months. Her mother (DMS-04.02), sister (DMS-04.03), current husband (DMS-04.05), and one child of the couple (DMS-04.01) accepted to participate in the study and the serology for HTLV-1/2 were negative for all them. DMS-04 reported receiving one bag of blood transfusion in 2020 due to blood loss in childbirth. She was also reported having an accident with others’ blood, having a tattoo, being diagnosed with herpes, and sharing personal objects like pliers. DMS-04.01 was born in 2008 and was not breastfed. The couple’s youngest daughter was born in 2020 and breastfed for 8 months. She tested for anti-HTLV-1/2 in the private health system and reported that the result was negative. Participant DMS-04 was referred to the medical service. During the consultation, the doctor consulted the previous result of the last antenatal screening and reported that she already had serology positive for anti-HTLV-1, and she reported being unaware of this result.A third blood donor candidate agreed to participate in the study (DMS-01), a 20-year-old female blood donor who had never donated blood before. The value obtained on the screening test in CMIA was a S/CO ratio value of 5.93. She reported as single in the research interview, but she reported in the blood bank questionnaire that was married. Her mother also agreed to participate in the study (DMS-01.01). Both DMS-01 and DMS-01.01 blood samples were negative HTLV-1/2 in our study. DMS-01 was referred to the medical service. She reported having rheumatologic symptoms, and other tests were ordered by the doctor.Some inconsistencies were found comparing the answers given to HEMOSUL before the donation and to research group during the interview, such as marital status and ethnicity. Regarding the other three confirmed HTLV blood donor candidates, the information available is based on the institutional database only Table . Of thes10, the indigenous population11, prisoners12, the Japanese community14, men who have sex with men15 and remnant quilombos16. The present study describes the seroepidemiological profile of blood donors from Mato Grosso do Sul state, Central Brazil. A study of blood donors from 27 Brazilian state capitals centers assessed the geographic distribution of HTLV screening prevalence rates between 1995 and 2000. It was observed a heterogeneous distribution of HTLV prevalence among the Brazilian regions, with higher rates in the Northern/Northeastern. In the state of Mato Grosso do Sul the prevalence estimated was 2.1/1,000, not considering confirmatory tests17. The prevalence found in the present study considering the initial screening tests (2.21/1000) indicates maintenance of the prevalence over more than 20 years between these studies. However, using confirmatory tests, the prevalence found in this study (0.017%—0.17/1000) shows to be a much lower value. Low prevalence was also observed in the pregnant population from Mato Grosso do Sul (0.13%)10. Other blood donor studies conducted in low prevalence regions in Brazil found rates of prevalence of 0.10%, 0.08%, 0.11%, 0.08%, 0.13%, and 0.15% in Araçatuba, Presidente Prudente, Serrana, Ribeirão Preto (São Paulo state), Manaus (Amazonas state) and in the state of Maranhão, respectively20.Previous studies on HTLV infection prevalence in the state of Mato Grosso do Sul have focused on specific groups, such as pregnant womenThe profile of eligible and inapt blood donor candidates for HTLV based on self-report is similar Table . In the The three positive individuals who agreed to participate in the study show some particularities. When accompanied in the medical service by the group, DMS-01 related some autoimmune symptoms, and her rheumatoid factor showed a positive result, which could have caused interference with HTLV screening, generating a false positive result in the confirmatory test realized on the blood bank. Fujirebio INNO-LIA HTLV I/II Score kit manufacturer’s instructions provide information that some infections may interfere with the analysis, as well as immunological changes such as autoimmune conditions.DMS-02 reported during the interview that, despite the referral to schedule an appointment received from HEMOSUL, she was unable to access the health service. She had been trying to schedule for almost a year. Since forwarding had no effect, this gap shows the urgent need to organize a referral system between the blood bank managed by the reference centers since the individual already presents laboratory results, a possible diagnosis, and the clinical condition of the same must be investigated.21. At the time, the child had 2 years old and did not present a seroconversion. The need for an improvement of maternal counseling about breastfeeding was already reported in the state11, demonstrating that infection patterns are not so well understood by health workers22 when they are expected that they should be able to properly provide counseling for positive patients about HTLV care and prevention.Participant DMS-06 was followed up throughout her entire antenatal period, but she related that she was not informed about her positive serology for anti-HTLV-1 during her last pregnancy. Her daughter was breastfed for 8 months, which could lead to mother-to-child transmission of HTLV through breastfeeding22. The default in marital status found in this study may be due to the omission of risk factors, such as possibly multiple partners. Regarding ethnicity, most of them reported as Brazilian caucasian. Other studies that used data from HEMOVIDA bring the information that Brazilian caucasian include brown and mixed race23, and there is no clear difference between them24. Brazilian Institute of Geography and Statistics (IBGE) has 5 options for self-declared race/color: white, black, brown, indigenous, or yellow25. We suggest a standardization following the IBGE pattern applied for the blood donor candidates.Regarding inconsistencies found in the answers given to HEMOSUL and the research group, it may be that some blood donors see the blood banks as a convenient place for free testing27. Then, the blood bags with S/CO < 4 may have had unnecessary disposal. Blood banks have no diagnostic purpose since the main objective is to avoid false negatives, then a high rate of false-positive results is expected in the blood bank screening tests due to low positive predictive value28, this implies an overestimation of the prevalence of HTLV infection if only a screening test is considered. Therefore, the algorithm of the tests influences the prevalence rate. In this study, we considered positive HTLV-confirmed result donors only. During the study period, the HEMOSUL blood bank performed confirmatory tests for HTLV on the positive samples in the screening. However, since May 2022, HEMOSUL has not performed confirmatory tests for HTLV. Furthermore, we must consider the impact of a false-positive result on the quality of life of a person without the confirmatory test to show him that he is uninfected. However, conducting confirmatory tests for HTLV is not mandatory for Brazilian blood bank services29.All individuals whose initial sample was anti-HTLV positive with a S/CO ratio < 11.79 was subsequently found to be HTLV negative. Although the manufacturer’s instructions of Abbott Architect rHTLV I/II assay indicate that samples with S/CO ratio ≥ 1 are considered reactive, previous studies suggest that the S/CO ratio < 4 excludes HTLV infection and the risk of blood transmissionConsidering that our data showed that the HTLV-confirmed samples were only 7.69% (6/78) of the initially positive samples and that there is no specific treatment for HTLV, identifying an infected blood donor candidate represents a great opportunity to block the transmission, consequently, the associated diseases, but this requires adequate counseling and patient management. This fact highlights the importance of retesting positive blood donors and/or submitting to a confirmatory test.We identified some limitations related to the HTLV infection prevalence in blood donors in the review literature. Most studies used initial blood bank screening data, which may result in an overestimation of prevalence due to the low cutoff value of the S/CO ratio. Another limitation of our study was the limited availability of behaviour pieces of information in the HEMOSUL database for each blood donor.In conclusion, our findings provide additional blood donors data for the prevalence of HTLV-1/2 in Brazil and highlight the need for a closer look at HTLV infection results in Brazilian blood banks, proposing confirmatory testing for those seropositive donors in screening assays and the need for adequate counseling and patient management for those confirmed HTLV infected individuals. We recommend implementing confirmatory tests at the blood bank or centralizing them within the public health system. Additionally, there is a need for a systematic approach to referring seropositive individuals to medical care, as access can be challenging.This study was approved by the Ethical Committee on Human Research of the Universidade Federal de Mato Grosso do Sul (CEP/UFMS) by protocol number CAAE: 50,022,721.6.0000.0021. All subjects or their legal guardians, in the case of individuals under age 18, gave their written informed consent to participate in the study at the time of sampling. All research was performed following relevant guidelines and regulations.A cross-sectional study was conducted among blood donors from Mato Grosso do Sul state (MS)—Central Brazil, between January 2021 to December 2021. Firstly, the data was obtained from databases, and samples from the blood collection from Mato Grosso do Sul Centro de Hematologia e Hemoterapia (HEMOSUL), which has 12 units in 10 cities . The inclusion criteria were to be a blood donor in the follow-up year. Then, the screened anti-HTLV seropositive individuals were invited to participate in the study with their family members to confirm the anti-HTLV seropositivity and to invite the family members of the case to participate. Inclusion criteria were: (i) a screening diagnosis for HTLV-1 infection; (ii) being a blood donor´s family member; (iii) having signed the informed consent form.Participants underwent an interview with a standardized questionnaire containing sociodemographic and sexual behavior information and provide blood samples. All subjects gave their written informed consent to participate in the study. In the case of individuals under age 18, informed written consent was obtained from themselves and their legal guardians.i2000SR equipment) for the presence of anti-HTLV-1/2 antibodies. It was considered positive when samples had a cut-off ≥ 1, following the manufacturer’s instructions. The grey zone considering a cut-off ranging from 0.9 to 1.0, is configured in the equipment by HEMOSUL. Positive samples were confirmed by HTLV-1/2 INNO-LIA assay (Fujirebio INNO-LIA HTLV I/II Score).In the follow-up year, the coordinating center of HEMOSUL screened all serum samples using chemiluminescent microparticle immunoassay (CMIA)  commercial kit for the presence of anti-HTLV-1/2 antibodies (Murex HTLV I + II—DiaSorin), following the manufacturer’s instructions. Positive samples were repeatedly tested and confirmed by HTLV-1/2 Western Blot (WB) assay (MP Diagnostics HTLV BLOT 2.4—Singapore). HTLV infection was defined as a repeatedly positive ELISA and positive WB . Categorical variables were presented with absolute and percentage frequency. Continuous variables were expressed as mean, standard deviation, median, and range.


Baicalin has been shown to promote spatial learning and neural regeneration, which might increase the differentiation of neural stem cells in Alzheimer’s disease (AD) rat models. We aimed to study the role of baicalin on neuronal pentraxin-1 (NPTX-1), neuronal pentraxin-2 (NPTX-2), and C-reactive protein (CRP) in AD model rats.The 30 male Sprague Dawley rats were divided into three groups: the control group, the AD model group, and the AD + baicalin group. Then, the Morris water maze was used to verify the effect of baicalin on the memory and spatial learning of rats. Immunohistochemistry and immunofluorescence were used to observe the expression of NPTX-1, NPTX-2, and CRP in brain tissue.P = 0.008) and had longer cross-platform times in the target quadrant (P = 0.015). In addition, the AD + baicalin group had significantly higher numbers of hippocampal neurons compared with the AD model group (P < 0.05). Baicalin also obviously decreased the apoptosis of neurons. Moreover, compared with the AD model group, the NPTX-1 and CRP expression in the AD + baicalin group was significantly reduced (P = 0.000) while the expression of NPTX-2 in the brain tissue of AD rats was significantly increased (P = 0.000).Compared with the AD model group, the AD rats treated with baicalin spent significantly less time finding escape latencies (Baicalin can play a therapeutic role by downregulating NPTX-1, upregulating NPTX-2, and downregulating CPR in AD model rats. It has been reported that immunological changes, oxidative stress, microvascular changes, inflammatory reactions, and excitotoxicity were the main mechanisms in the development of AD . AlthougThe prominent role of pentraxins (PTXs), including neuronal pentraxin-1 (NPTX-1), neuronal pentraxin-2 (NPTX-2), and C-reactive protein (CRP) in synaptic plasticity raises the possibility that its expression may be altered in AD that are characterized by synaptic dysfunction and loss . PreviouScutellaria baicalensis Georgi. [Recently, baicalin has been shown to promote spatial learning and neural regeneration , which m Georgi. . Jin et  Georgi.  indicate Georgi. . NPTX1, Thus, in our study, we evaluated the effect of baicalin on AD rats using Morris water maze (MWM). Then, the immunohistochemistry and immunofluorescence were used to observe the expression of NPTX-1, NPTX-2, and CRP in brain tissue. Our results may offer a theoretical basis for the use of baicalin, the active components of which promote nerve regeneration.22.1Baicalin (purity >99%) was supplied by Shanghai yuanye Bio-Technology Co., Ltd . Amyloid β-protein, Fragment1-40 (Aβ1-40)  was attenuated to 5 g/L with sterile double-distilled water, and then incubated in an incubator (37°C) for 7 days. After that, Aβ1-40 was stored at −20°C.2.2A total of 30 healthy Sprague Dawley (SD) male rats (weighing 250–300 g) were obtained from the Animal Center of First Hospital affiliated to Harbin Medical University . The SD rats were raised under the barrier system of room temperature and humidity (40–50%), and experimental conditions were kept according to the SPF standard.n = 10 per group) for anesthesia without signs of peritonitis. Rat brains were fixed on a stereoscopic instrument, and the hair and scalp were removed to make the cranium expose. The hippocampus was injected unilaterally with 2 μL (10 µg) Aβ1-40 solution using a micro-syringe in 5 min. The control group was treated with an equal volume of sterile normal saline. After surgery, dental clay was used to seal the holes and the scalp was sutured and treated with a disinfectant to avoid infection [The 30 SD rats were randomly classified into the control group, the AD model group, and the AD + baicalin group , was first applied by Morris (1984) to assess memory function and spatial learning which normally used visual cues . We usedThe spatial probe test was used to detect changes within the spatial memory of the rats. This test was conducted on the second day after the place navigation test, during which time the platform was removed. The rats were placed into the water at a random entry point within the quadrant corresponding to the platform. The rats were allowed to swim freely in the water for 90 s. Then, we recorded the time that rats stayed in the quadrant corresponding to the platform.2.4Chloral hydrate was used to anesthetize animals at a dose of 300 mg/kg. After fixation, the brains were removed, and then the rats immediately were euthanized with an injection of sodium pentobarbital at a dose of 150 mg. When the rat’s breathing stops and the heart stops beating, it is determined to be dead. The brain was dehydrated by gradient alcohol, and embedded in paraffin for immunohistochemical analysis, HE staining , and immunofluorescence. The paraffin-embedded tissue was cut into 3 µm-thick sections and paraffin was removed by gradient ethanol. Brain sections were stained with hematoxylin for 1–2 min and eosin for 30–60 s. After that, the sections were dehydrated with ethanol, sealed with neutral gum, and cleared with xylene . After f2.5Sections of paraffin-embedded brain slices were deparaffinized and rehydrated before blocking non-specific antigen binding with 5% bovine serum albumin . The following primary antibodies were used: NPTX-1 , NPTX-2 , and CRP . Goat anti-rat/rabbit  polymer enhancer and goat-resistant rabbit/rat IgG-conjugated polymer were added to each tissue section. Afterward, the immunostained sections were developed in diaminobenzidine  and counterstained with a weak solution of hematoxylin solution. The stained tissue slices were visualized on a microscope .NPTX-1, NPTX-2, and CRP. The luciferin antibody (100 µL) was added to each stained section. After incubating the sections in the dark at room temperature for 1 h, a drop of 4′,6-diamindino-2-phenylindole  staining agent was added to each tissue section. An anti-quenching agent was added to seal the sections. The staining results were observed under a fluorescence microscope.The immunofluorescence staining protocol on the first day was the same as the immunohistochemical staining protocol, which was performed to evaluate the localization of 2.6The radioimmunoprecipitation assay buffer was used to isolate the total protein, and then it was quantified using bicinchoninic acid assay . The total protein (20 μg) was added into every well, and then transferred onto polyvinylidene fluoride membrane . The primary antibody including Bax , caspase-3 , and actin  were incubated with membrane at 4°C overnight. Secondary goat anti-rabbit antibody  and goat anti-mouse IgG  were incubated with membrane at room temperature for 2 h. The protein bands were visualized and the images were analyzed through chemiluminescence meter .2.7P value <0.05 was deemed statistically significant.The experimental data positioning navigation and space exploration time from MWM were recorded and analyzed using one-way ANOVA method in SPSS software 22.0 . The data were exhibited as mean ± standard deviation. Then, the immunohistochemical staining results of NPTX-1, NPTX-2, and CRP were semi-quantitatively analyzed, and ten fields of view in and around the hippocampus were selected for high-magnification imaging through an optical microscope, and the integrated optical density (IOD) value of each image was measured using ImageJ digital software. The average value of the 10 IOD values obtained for each slice represents the average IOD value of the slice. The IOD values obtained from the brain slices of mice in different groups are expressed as mean ± SD. Meanwhile, the immunofluorescence results to evaluate the gene localization were measured using ImageJ digital software. The formula was as follows: average fluorescence intensity (Mean) = sum of fluorescence intensity in this area (IntDen)/area of this area (Area). The difference between the groups was analyzed using one-way analysis of variance. A Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. Approval was obtained from the Ethics Committee of The First Hospital of Harbin Medical University  2017-003) and conducted in strict accordance to the standard of the Guide for the Care and Use of Laboratory Animals published by the Ministry of Science and Technology of the People’s Republic of China in 2006.33.1The MWM test was used to assess the memory and spatial learning of rats. It is on the sixth day that the memory and spatial learning of rats was improved in AD + baicalin group when compared with AD group . In addi3.24 cell/mm3 vs 4.132 × 104 cell/mm3, P < 0.05). However, the mean number of hippocampal neurons in the AD + baicalin group was significantly higher than the AD model group  , which were consistent with the results of HE staining.In the controls, no obvious cell deletion of the pyramidal cells was found. The morphology of the cells was complete, the nucleus was clear, and the cells were round or quasi-round . However < 0.05) . Moreove3.3P = 0.000)  . The flu= 0.000) ; Table 3P = 0.033) (P = 0.021)  ; Table 3= 0.033) . In addi= 0.021) . The flu= 0.021) ; Table 44Previous studies demonstrated that baicalin and PTXs play the vital role in the development of AD. Previous studies did not show that baicalin impacts the expression of NPTX-1, NPTX-2, and CRP. Baicalin is a vital element of traditional Chinese, which can effectively protect against AD pathology . HoweverBesides, NPTX-1 and NPTX-2 can participate in the formation, homeostasis, and plasticity of synaptic, and other processes alone or jointly, which play an important role in the process of clearing the synaptic debris generated during synaptic remodeling . Both thSchmidt et al.  found th5In conclusion, baicalin may improve memory function and learning ability in a rat model of AD. We believe that the therapeutic actions of baicalin are linked with its ability to downregulate NPTX-1 and CPR, and upregulate NPTX-2 in a rat AD model.ADAlzheimer’s diseaseCRPC-reactive proteinNPTXneuronal pentraxinSAPamyloid substanceSDSprague DawleySDstandard deviation


We explored the potential of targeting the enzyme cytochrome P450 (CYP450) in colorectal cancer (CRC) using lopinavir/ritonavir. Various experimental methods in a preclinical phase were employed to assess the effects of lopinavir/ritonavir on CRC. The study found that inhibiting CYP450 reduced cell proliferation, induced cell death, and suppressed cell migration. Lopinavir/ritonavir also inhibited tumor growth and fibrosis. These results suggest that targeting CYP450 with lopinavir/ritonavir has therapeutic potential in CRC and highlight the need for further research in this area.Cytochrome P450 (CYP450) enzyme has been shown to be expressed in colorectal cancer (CRC) and its dysregulation is linked to tumor progression and a poor prognosis. Here we investigated the therapeutic potential of targeting CYP450 using lopinavir/ritonavir in CRC. The integrative systems biology method and RNAseq were utilized to investigate the differential levels of genes associated with patients with colorectal cancer. The antiproliferative activity of lopinavir/ritonavir was evaluated in both monolayer and 3-dimensional (3D) models, followed by wound-healing assays. The effectiveness of targeting CYP450 was examined in a mouse model, followed by histopathological analysis, biochemical tests , and RT-PCR. The data of dysregulation expressed genes (DEG) revealed 1268 upregulated and 1074 down-regulated genes in CRC. Among the top-score genes and dysregulated pathways, CYPs were detected and associated with poor prognosis of patients with CRC. Inhibition of CYP450 reduced cell proliferation via modulating survivin, Chop, CYP13a, and induction of cell death, as detected by AnnexinV/PI staining. This agent suppressed the migratory behaviors of cells by induction of E-cadherin. Moreover, lopinavir/ritonavir suppressed tumor growth and fibrosis, which correlated with a reduction in SOD/thiol levels and increased MDA levels. Our findings illustrated the therapeutic potential of targeting the CYP450 using lopinavir/ritonavir in colorectal cancer, supporting future investigations on this novel therapeutic approach for the treatment of CRC. According to recent estimates, colorectal cancer (CRC) ranks as the third leading cause of cancer-related fatalities . AccordiThe cytochrome P450 (CYP) superfamily is responsible for the catalytic oxidation of several substances . RecentlFor this study, lopinavir/ritonavir (200/50 mg) and 5-FU were purchased from Abbott C and Ebewe, respectively. RPMI 1640 medium, DMEM, FBS, streptomycin (50 g/mL), and penicillin (50 IU/mL) were all obtained from Betacell in Tehran, Iran. Sigma-Aldrich provided further chemical lab materials .2. The cells were checked under a microscope to confirm confluency of 80–90%, then cell passaging was performed using trypsin-EDTA. All procedures were according to ATCC instructions.CT-26 and SW-480 cell lines were obtained and certified by the National Cell Bank of Iran. All of the cultured media consisted of RPMI or DMEM, with 10% FBS and 1% streptomycin/penicillin. The culture condition was maintained at 37 °C with a 5% COTwenty-two colorectal cancer (CRC) tumors and their respective adjacent normal margins, validated by pathologists, were included in the study. Treatment for all eligible patients was administered at Omid Hospital of MUMS , and the patients had not received chemotherapy before. Their clinicopathological characteristics are described in The antiproliferative effect of lopinavir/ritonavir both alone and in combination with 5-FU was estimated via MTT assay after 24 h treatment. CT-26 and SW-480 cells were incubated in a 96-well plate and treated with various concentrations of lopinavir/ritonavir (1–500 μM) as well as its combination with 5-FU at a constant IC50 concentration of 5-FU. Then, the MTT test was assessed .3 CT-26 cells/mL in a mixture of DMEM/F12 and GlutaMAX-I in a ratio of 1:1 were seeded in 96-well agarose-coated plates to produce multicellular spheroids. After spheroid formation, cells were treated with the IC50 dose in the complete medium for 10 days. The volume (V) of the spheroid was determined using the geometric mean of its perpendicular diameters (D = (Dmax + Dmin)/2) after the spheroid diameter was measured using ImageJ. V = (4/3) × π(D/2)3  = A/1.65 × 105) .The total thiol levels were measured using the Ellman method. When sulfhydryl groups bonded to carbon react with Ellman’s reagent, 5,5′-dithiobis, a yellow complex with a peak absorbance at 412 nm, is formed. To measure the absorbance, 50 μL of supernatant from each sample was mixed with 1 mL of EDTA buffer and compared to the absorbance of the buffer alone (A1) at 412 nm. After 15 min, the sample absorbance was measured once more, and A2 was labeled after 20 μL of DTNB solution had been added to A1. DTNB absorbance was used as the blank (B). The following equation was used: = (A2 − A1 − B) × 1.07/0.05 × 13.6 .The method of Madesh and Balasubramanian was utilized to measure superoxide dismutase (SOD) activity. The method depends on the dependent suppression of MTT to formazan and SOD generation from pyrogallol auto-oxidation. The process was stopped with the addition of DMSO. The supernatant was added to a 96-well plate. After 5 min, DMSO was introduced, and the plate was subsequently analyzed using a microplate reader at 570 nm. One unit of superoxide dismutase (SOD) activity was defined as the amount of protein necessary to prevent a 50% drop in MTT.2O2 with phosphate buffer (pH = 7) (C buffer). In addition, 650 microliters of pH-7.0 phosphate buffer were used as a solution blank. The cuvette was filled with sample homogenates and C buffer before measurements. A spectrophotometer was used to measure the reduction in absorbance for 5 min at a wavelength of 240 nm.The solution to be used to measure the catalase activity was prepared by mixing 100 μL of HThe orientation of lopinavir/ritonavir in the target proteins’ active site was assessed through MOE web-based. We carried out the data analysis by ChemDraw Ultra 7.0 to draw the structure of lopinavir/ritonavir, which was then subjected to energy minimization by MOE. The crystal structure of proteins was obtained from the RCSB Protein Data Bank. The GBVI/WSA dG scoring method was utilized to evaluate the concluding scores ,21. The p-values < 0.05 and Log2-fold change |1.5| >, with statistical significance considered at p < 0.05.We undertook gene expression profiling on 22 cases of colorectal cancer (CRC) with the SRA study ID. We utilized the FeatureCounts package in Linux (ubuntu 20.4 LTS). We then normalized the data and analyzed the dysregulation expressed genes (DEGs) using the DESeq2 package (v.1.20) in R software (v.3.6). Next, we compared two conditions: CRC patients with advanced-stage versus normal cases. We used DESeq2 and ClusterProfiler packages to identify up- and down-regulated genes, which were then analyzed for Gene Set Enrichment using the AnnotationHub package to reveal Disease Ontology, Network of Cancer Gene, Gene Ontology, and KEGG Pathways. In addition, gene ontology, which includes biological processes, molecular function, and cellular components, was analyzed using Enrich r to determine the mutual roles of accumulated genes in the cell. To ensure reliable results, we used special criteria, including p-value of <0.05 was considered significant. At least two separate experiments were carried out in triplicate for each experiment to ensure the reliability of the results.Results were expressed as mean ± standard error of the mean (SEM). The graphs were created using Graph-Pad Software version 8 and all of the data were analyzed with SPSS version 20 software. Statistical analyses included one-way ANOVA and Tukey–Kramer tests. A After analyzing the dysregulation expressed genes (DEGs), we found that 1268 genes were up-regulated and 1074 genes were down-regulated . The topThe results of the MTT assay showed a dose-dependent inhibitory activity of lopinavir/ritonavir on cell viability with an IC50 of 51.52 μM and 77.17 μM in CT-26 and SW-480 cells, respectively. Moreover, combining lopinavir/ritonavir with 5-FU had IC50 values of 38.77 μM and 59.24 μM in CT-26 and SW-480 cells A. The inTo examine the inhibitory effect of lopinavir/ritonavir on cell migration, the scratch assay was used. CT-26 and SW-480 cell were treated and evaluated in 0, 48, and 72 h. The results indicated that lopinavir/ritonavir prevents cell migration in both cell lines . To inveTo further evaluate the results from the in vitro study, an animal model of CRC was developed. As shown in To assess the effect of lopinavir/ritonavir on oxidant/antioxidant balance, we also measured the amounts of thiol, CAT, SOD, and MDA in the homogenized tumor samples. In addition, we carried out an in-silico analysis  and inveFinally, within We have presented the first proof-of-concept preclinical finding on the potential therapeutic value of lopinavir/ritonavir in the treatment of CRC. The activation and detoxification of a wide range of chemotherapeutic agents are mediated by cytochrome P450 (CYP) enzymes belonging to families one to three . CYP3A4,The cytotoxic effects of HIV-PIs have stimulated research on the repurposing of these drugs as anticancer medications . Less reThe antitumor efficacy of lopinavir/ritonavir was assessed in colon cancer cells. Our results revealed that lopinavir/ritonavir, as well as its combination with 5-FU, exhibited dose-dependent inhibition of cell growth in CT-26 and SW-480 cell lines. Interestingly, these findings were particularly evident in 3D models, which are more representative of tumor architecture.The levels of cyclin D1 and survivin were also evaluated, and we found that lopinavir/ritonavir treatment resulted in a significant decrease in the mRNA level of survivin, which is in accordance with prior research showing the growth-inhibiting effects of PIs via downregulation of survivin and induction of apoptosis . It has Our results are also in line with previous reports demonstrating the anti-migratory effects of PIs in cancer cell lines . The obsOur in vivo studies showed that lopinavir/ritonavir and its combination with 5-FU inhibited the formation of tumor growth. Moreover, the impact of lopinavir/ritonavir on the oxidant/antioxidant balance in tumor tissue was evaluated by quantifying total thiol content, CAT activity, SOD activity, and MDA levels in the samples ,50,51. SOur study suggests that lopinavir/ritonavir has promising therapeutic potential for the treatment of colorectal cancer. We found that it effectively inhibits cell growth and migration in CRC cell lines. Our findings also suggest that lopinavir/ritonavir may exert its antitumor effects by inducing apoptosis, as well as suppressing the migratory behavior of CRC cells. Further clinical studies are needed to confirm its clinical efficacy and safety.